The use of RNA offers an attractive alternative to DNA in order to circumvent the potential safety risks connected with the therapeutic use of DNA. In vitro-transcribed RNA (IVT-RNA) is of particular interest in therapeutic approaches. The advantages of a therapeutic use of RNA include transient expression and a non-transforming character. RNA does not need to enter the nucleus in order to be expressed and moreover cannot integrate into the host genome, thereby eliminating the risk of oncogenesis. When used for vaccination, injection of RNA can induce both cellular and humoral immune responses in vivo. However, the use of RNA for clinical applications is greatly restricted especially by the short half life of RNA.
IVT vectors may be used in a standardized manner as template for in vitro transcription. Such IVT vectors may have the following structure: a 5′ RNA polymerase promoter enabling RNA transcription, followed by a gene of interest which is flanked either 3′ and/or 5′ by untranslated regions (UTR), and a 3′ polyadenyl cassette containing A nucleotides. Prior to in vitro transcription, the circular plasmid is linearized downstream of the polyadenyl cassette by type II restriction enzymes (recognition sequence corresponds to cleavage site). The polyadenyl cassette thus corresponds to the later poly(A) sequence in the transcript.
The 3′ poly(A) sequence of RNA is important for nuclear export, RNA stability and translational efficiency of eukaryotic messenger RNA (mRNA). The 3′ poly(A) sequence is shortened over time and if short enough, the RNA is degraded enzymatically.
We have previously demonstrated that a 3′ poly(A) sequence with a length of 120 nucleotides (A120) has a predominant effect on RNA stability and translational efficiency and thus, is beneficial for all-over RNA efficacy.
However, it has been observed that the DNA sequence encoding the 3′ poly(A) sequence (3′ polyadenyl cassette), i.e. a stretch of consecutive dA:dT base pairs, is subjected to shortening in some bacterial subclones, when propagated in E. coli. Accordingly, before producing the plasmid DNA as the starting material for in vitro transcription a large number of bacterial clones has to be tested, e.g., by determining the length of the 3′ polyadenyl cassette by suitable restriction analysis, to obtain a single clone with a 3′ polyadenyl cassette of the correct length encoding a 3′ poly(A) sequence of the correct length.
It was the object of the present invention to find a 3′ polyadenyl cassette which shows constant propagation with the coding plasmid DNA in E. coli and which encodes a 3′ poly(A) sequence maintaining the effects with respect to supporting RNA stability and translational efficiency.
This object is achieved according to the invention by the subject matter of the claims.
According to the invention, it was found that a disruption of the 3′ polyadenyl cassette (poly(dA:dT) region) by a 10 nucleotide random sequence, with an equal distribution of the 4 nucleotides (linker), has only minor influence on functionality of the encoded RNA but increases the stability of the 3′ polyadenyl cassette in E. coli. Further, neither the sequence nor the position of the linker within the 3′ poly(A) sequence resulted in a reduction of translational efficiency and stability of the in vitro transcribed RNA (IVT RNA).
For stability testing of the IVT vector region encoding the 3′ poly(A) sequence, the SIINFEKL peptide (SEQ ID NO: 1) was cloned upstream of the poly(dA:dT) region. This construct showed a poly(dA:dT) instability (i.e. percentage of clones upon propagation with shortened poly(dA:dT)) of 50-60%. Detailed analysis, using the described restriction analysis method identified the region at position 30-50 as being particular sensitive to shortening of the poly(dA:dT) stretch. Introduction of a 10 nucleotide random sequence in this sensitive region led to an increase of the poly(dA:dT) stability. Constructs with 30 or 40 adenosine nucleotides, followed by the linker sequence and another 70 or 60 adenosines (A30L70 and A40L60) respectively, resulted in an poly(dA:dT) instability of only 3-4% in E. coli. Results were confirmed by testing the constructs in several different E. coli strains.
Functionality of IVT RNA encoded by DNA carrying the stabilized poly(dA:dT)-tails was tested in different assays. Electroporation of the IVT RNA in somatic cell lines but also in immune cells such as immature dendritic cells showed no difference in translational capacity compared to the A120 over a time period of 72 hours. Injection of luciferase encoding IVT RNA into mice confirmed an equal protein translation independent of the inserted type of the 3′ poly(A) sequence.
An impact on the immunological response of the different 3′ poly(A) sequences was analyzed by comparison of the amount of antigen-specific CD8+ T-cells upon injection of SIINFEKL (SEQ ID NO: 1) IVT RNA. The experiments revealed no difference between the A120 and its stabilized versions A30L70 and A40L60.
Taken together, we show that the insertion of a 10 nucleotide random sequence between position 30 and 50 of a poly(dA:dT) region results in a more than 10-fold sequence stabilization in E. coli. The corresponding modified 3′ poly(A) sequence of the RNA transcribed from the template DNA has the same functionality, i.e. stability and translational efficiency in vivo and in vitro as the classical A120. Additionally the immunological response is not altered by the use of a modified poly(A) sequence.